EP0100522B1 - Protéine PP17, procédé pour sa concentration et son obtention ainsi que son application - Google Patents

Protéine PP17, procédé pour sa concentration et son obtention ainsi que son application Download PDF

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Publication number
EP0100522B1
EP0100522B1 EP83107375A EP83107375A EP0100522B1 EP 0100522 B1 EP0100522 B1 EP 0100522B1 EP 83107375 A EP83107375 A EP 83107375A EP 83107375 A EP83107375 A EP 83107375A EP 0100522 B1 EP0100522 B1 EP 0100522B1
Authority
EP
European Patent Office
Prior art keywords
protein
proteins
acetyl
isolating
human
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired
Application number
EP83107375A
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German (de)
English (en)
Other versions
EP0100522A2 (fr
EP0100522A3 (en
Inventor
Hans Dr. Bohn
Wilhelm Winckler
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Siemens Healthcare Diagnostics GmbH Germany
Original Assignee
Behringwerke AG
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Behringwerke AG filed Critical Behringwerke AG
Priority to AT83107375T priority Critical patent/ATE38388T1/de
Publication of EP0100522A2 publication Critical patent/EP0100522A2/fr
Publication of EP0100522A3 publication Critical patent/EP0100522A3/de
Application granted granted Critical
Publication of EP0100522B1 publication Critical patent/EP0100522B1/fr
Expired legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4715—Pregnancy proteins, e.g. placenta proteins, alpha-feto-protein, pregnancy specific beta glycoprotein
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
    • Y10S530/806—Antigenic peptides or proteins
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
    • Y10S530/827—Proteins from mammals or birds
    • Y10S530/85—Reproductive organs or embryos
    • Y10S530/851—Placenta; amniotic fluid

Definitions

  • the invention relates to a new protein (PP 17 ), a method for its enrichment and extraction from the extract of human placentas, and its use.
  • the present invention describes the isolation and characterization of a new soluble placenta protein called PP 17 .
  • An average of 2.5 mg of this protein can be extracted from an adult human placenta (600 g) with physiological saline.
  • Extracts from other human organs do not contain this protein or contain it in a much lower concentration.
  • PP 17 does not normally occur in the serum of other human body fluids either, or only in traces of 1 mg / l).
  • the electrophoretic mobility was examined in the micromodification with the Microzone R 200 device from Beckman Instruments on cellulose acetate films (Sartorius) using sodium diethylbarbiturate buffer, pH 8.6.
  • the isoelectric point was determined using a column (440 ml) from LKB, Sweden.
  • the so-called ampholin (R) mixture had a pH range of 4.0 to 6.0 when examining the glycoprotein.
  • the sedimentation coefficient was determined in an analytical ultracentrifuge from Beckman (Spinco apparatus, model E) at 60,000 rpm in double selector cells using the UV scanner technology at 280 nm. A 0.05 M phosphate buffer (pH 6.8) containing 0.2 mol / l NaCl was used as the solvent. The protein concentration was adjusted to an optical density of about 3. The sedimentation coefficient was converted to water at 20 ° C.
  • the substance was dissolved in 0.10% (g / 100 ml) in distilled water which had been adjusted to pH 7.0 by adding ammonium bicarbonate.
  • the analysis of the carbohydrates was carried out as follows: After hydrolysis of the glycosidic bonds, the released neutral sugars were separated as borate complexes via an anion exchange column (YC Lee et al., Anal. Biochem. 27, (1969) 567), in the eluate by Addition of Cu (I) bicinchoninate reagent (K. Mopper and M. Gindler, Anal. Blochem., 56, (1973), 440) stained and quantified using rhamnose as an internal standard. The aminosugars were detected and determined by their reaction with ninhydrin. The neuraminic acid content was determined using the Warren method (Methods in Enzymology, Vol. VI, 1963) 463-465).
  • the amino acid analysis was carried out according to S. Moore, D.H. Spackman, W.H. Stone, anal. Chem., 30, (1958), 1185, using the Multichrom B liquid chromatograph from Beckman. 1/2 cystine was obtained after oxidation of the proteins with performic acid (S. Moore et al., Anal. Chem., 30, (1958) 1185,) and subsequent chromatography (S. Moore, J. Biol. Chem., 238, ( 1963) 235) as cysteic acid. The tryptophan content was determined using the direct photometric determination according to H. Edelhoch, Biochemi, 6, (1967) 1948.
  • PP 17 has the following properties, which can be used in a process for its isolation by taking appropriate measures:
  • the invention relates to a process for obtaining the PP 17 , characterized in that the extract from human placentas is fractionated using the above properties.
  • the PP 17 can be isolated by an appropriate combination of the measures mentioned, which bring about an enrichment of the PP 17 or a separation of this protein from other proteins.
  • the subject matter of the present invention can be seen in the individual steps for enriching the PP 17 and in the method for cleaning the PP 17 which results from combining the measures for enrichment.
  • the example describes the isolation of PP 17 using the immunoadsorption method.
  • the aqueous extract from human placentas could be used directly for immunoadsorption. Since the concentration of PP 17 in the extract is relatively low, it is advisable to first enrich the protein PP 17 specifically by pre-fractionating the extract.
  • the immunoadsorption step could also be replaced by using other separation methods, for example by preparative electrophoresis and isoelectric focusing after using dialysis against an acid buffer of pH 2-4, preferably 0.5 M glycine-HCl (pH 2.5) .
  • immunochemical methods can also be used to detect PP 17, for example in a fraction from a separation operation, since PP 17 has antigenic properties. Specific antibodies are formed when animals are immunized with this protein.
  • An antiserum that can be used for this purpose can be obtained as follows:
  • PP 17 When fractionating the placenta extract with 2-ethoxy-6,9-diaminoacridine lactate and ammonium sulfate according to H. Bohn, (Arch. Gynäkol., 210, (1971), 440), PP 17 mainly goes into piazza fraction 111. This fraction becomes 14 Dialyzed for hours against a 0.5 M glycine-HCl buffer (pH 2.5), then neutralized and dialyzed against physiological saline. By immunizing rabbits with this fraction, a polyvalent antiserum is obtained with which PP 17 can be detected. This antiserum may be made specific largely 17 by absorption with normal human serum and those placenta fractions PP 17 is not included, to the antigen PP. This specific antiserum can serve on the one hand for the immunological detection of PP 17 and on the other hand for the production of an immunoadsorbent which can be used for the enrichment and isolation of PP 17 .
  • monospecific antisera can then be prepared directly by immunizing animals using known methods.
  • Figure 1a shows the immunological reaction of PP 17 with a specific rabbit antiserum after separation in an electric field in agar-containing gel.
  • Figure 1 b shows the separation of the proteins of the serum, made visible by their immune reaction with an antiserum from rabbits against human serum (HS).
  • HS human serum

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Genetics & Genomics (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Reproductive Health (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Toxicology (AREA)
  • Zoology (AREA)
  • Pregnancy & Childbirth (AREA)
  • Gynecology & Obstetrics (AREA)
  • Immunology (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Claims (5)

1. Protéine PP17, caractérisée par
a) une mobilité électrophorétique dans le domaine compris entre les globulines β1 et a2,
b) un point isoélectrique de 5,25 ± 0,20,
c) un coefficient de sédimentation S0 20,w de 2,7 ± 0,1 S,
d) une masse moléculaire déterminée dans un gel de polyacrylamide contenant du docécylsulfate de sodium (SDS) de 38 000 ± 2 000,
e) un coefficient d'extinction E1%1cm (280 nm) de 8,5 ± 0,4, et
f) une teneur en hydrates de carbone de 2,1 ± 0,9 %, contenant (0,3 ± 0,2 % de mannose, 0,4 ± 0,2 % de galactose, 0,2 ± 0,1 % de xylose, 1,0 ± 0,3 % de N-acétyl-glucosamine et 0,2 ± 0,1 % d'acide N-acétyl-neuraminique).
2. Procédé de préparation de la protéine selon la revendication 1, caractérisé en ce qu'on soumet un extrait de placentas humains qui contient une protéine présentant les paramètres indiqués dans la revendication 1 à une combinaison appropriée de plusieurs mesures opératoires connues pour l'isolement de protéines, en obtenant à chaque fois la matière qui contient la protéine avec les propriétés indiquées, et en obtenant la protéine.
3. Utilisation de la protéine suivant la revendication 1 pour la préparation d'anticorps spécifiques contre cette protéine.
4. Utilisation de la protéine dans des méthodes immunologiques de détection et le dosage de cette protéine.
EP83107375A 1982-07-30 1983-07-27 Protéine PP17, procédé pour sa concentration et son obtention ainsi que son application Expired EP0100522B1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AT83107375T ATE38388T1 (de) 1982-07-30 1983-07-27 Neues protein (pp17), verfahren zu seiner anreicherung und gewinnung sowie seine verwendung.

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE3228503 1982-07-30
DE19823228503 DE3228503A1 (de) 1982-07-30 1982-07-30 Neues protein (pp(pfeil abwaerts)1(pfeil abwaerts)(pfeil abwaerts)7(pfeil abwaerts)), verfahren zu seiner anreicherung und gewinnung sowie seine verwendung

Publications (3)

Publication Number Publication Date
EP0100522A2 EP0100522A2 (fr) 1984-02-15
EP0100522A3 EP0100522A3 (en) 1986-05-21
EP0100522B1 true EP0100522B1 (fr) 1988-11-02

Family

ID=6169725

Family Applications (1)

Application Number Title Priority Date Filing Date
EP83107375A Expired EP0100522B1 (fr) 1982-07-30 1983-07-27 Protéine PP17, procédé pour sa concentration et son obtention ainsi que son application

Country Status (7)

Country Link
US (1) US4468345A (fr)
EP (1) EP0100522B1 (fr)
JP (1) JPS5944321A (fr)
AT (1) ATE38388T1 (fr)
AU (1) AU555759B2 (fr)
CA (1) CA1213212A (fr)
DE (2) DE3228503A1 (fr)

Families Citing this family (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE3230996A1 (de) * 1982-08-20 1984-02-23 Behringwerke Ag, 3550 Marburg Neues protein (pp(pfeil abwaerts)1(pfeil abwaerts)(pfeil abwaerts)3(pfeil abwaerts)), verfahren zu seiner anreicherung und gewinnung sowie seine verwendung
DE3315000A1 (de) * 1983-04-26 1984-10-31 Behringwerke Ag, 3550 Marburg Gewebeprotein pp(pfeil abwaerts)4(pfeil abwaerts), verfahren zu seiner gewinnung sowie seine verwendung
DE3334405A1 (de) * 1983-09-23 1985-04-04 Behringwerke Ag, 3550 Marburg Membranassoziierte proteine (mp(pfeil abwaerts)2(pfeil abwaerts)), verfahren zu ihrer gewinnung sowie ihre verwendung
DE3338480A1 (de) * 1983-10-22 1985-05-02 Behringwerke Ag, 3550 Marburg Neues protein pp(pfeil abwaerts)2(pfeil abwaerts)(pfeil abwaerts)0(pfeil abwaerts), verfahren zu seiner gewinnung sowie seine verwendung
DE3404563A1 (de) * 1984-02-09 1985-08-14 Behringwerke Ag, 3550 Marburg Gewebeprotein pp(pfeil abwaerts)1(pfeil abwaerts)(pfeil abwaerts)9(pfeil abwaerts), verfahren zu seiner gewinnung sowie seine verwendung
DE3410694A1 (de) * 1984-03-23 1985-10-03 Behringwerke Ag, 3550 Marburg Gewebeprotein pp(pfeil abwaerts)2(pfeil abwaerts)(pfeil abwaerts)1(pfeil abwaerts), verfahren zu seiner gewinnung sowie seine verwendung
DE3418888A1 (de) * 1984-05-21 1985-11-21 Behringwerke Ag, 3550 Marburg Gewebeprotein pp(pfeil abwaerts)1(pfeil abwaerts)(pfeil abwaerts)8(pfeil abwaerts), verfahren zu seiner gewinnung sowie seine verwendung
JPH0689014B2 (ja) * 1986-01-21 1994-11-09 興和株式会社 トロンビン結合性物質およびその製法
AU610916B2 (en) * 1987-08-04 1991-05-30 Meguro Institute Co., Ltd. Human placenta-derived thermostable cell growth factor and a process for production thereof
US5252210A (en) * 1992-09-15 1993-10-12 Wagner Spray Tech Corporation Paint intake filter guard
US5968477A (en) 1994-01-24 1999-10-19 Neorx Corporation Radiolabeled annexin conjugates with hexose and a chelator
US20030220233A1 (en) * 1994-01-24 2003-11-27 Neorx Corporation Radiolabeled annexins
CA2190727C (fr) * 1994-05-19 2006-07-18 Sudhakar Kasina Ligands d'atomes donneurs d'azote et de soufre pontes et substitues par amines aromatiques, utilises en imagerie
US6005083A (en) 1997-03-28 1999-12-21 Neorx Corporation Bridged aromatic substituted amine ligands with donor atoms

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0009715A1 (fr) * 1978-09-29 1980-04-16 BEHRINGWERKE Aktiengesellschaft Protéine ubiquitaire de tissu PP8 et procédé pour sa préparation

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US2042430A (en) * 1934-06-18 1936-05-26 Lakeland Foundation Pregnancy antigen
DE2221261A1 (de) * 1972-04-29 1973-11-15 Behringwerke Ag Ap-glykoproteine und verfahren zu ihrer isolierung
DE2640387C3 (de) * 1976-09-08 1981-01-22 Behringwerke Ag, 3550 Marburg Gewebespezifisches Protein und Verfahren zu dessen Herstellung
DE2952792A1 (de) * 1979-12-31 1981-07-02 Behringwerke Ag, 3550 Marburg Neues protein (pp(pfeil abwaerts)15(pfeil abwaerts)) mit immunsuppressiver wirkung
DE3013724A1 (de) * 1980-04-10 1981-10-15 Behringwerke Ag, 3550 Marburg Neues protein pp (pfeil abwaerts)9(pfeil abwaerts), verfahren zu seiner anreicherung und gewinnung sowieseine verwendung
DE3109629A1 (de) * 1981-03-13 1982-09-23 Behringwerke Ag, 3550 Marburg "neues protein (pp(pfeil abwaerts)1(pfeil abwaerts)(pfeil abwaerts)6(pfeil abwaerts)), verfahren zu seiner anreicherung und gewinnung sowie seine verwendung"

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0009715A1 (fr) * 1978-09-29 1980-04-16 BEHRINGWERKE Aktiengesellschaft Protéine ubiquitaire de tissu PP8 et procédé pour sa préparation

Also Published As

Publication number Publication date
JPH0354679B2 (fr) 1991-08-20
AU555759B2 (en) 1986-10-09
EP0100522A2 (fr) 1984-02-15
EP0100522A3 (en) 1986-05-21
US4468345A (en) 1984-08-28
CA1213212A (fr) 1986-10-28
DE3228503A1 (de) 1984-02-02
DE3378368D1 (en) 1988-12-08
JPS5944321A (ja) 1984-03-12
AU1745383A (en) 1984-02-02
ATE38388T1 (de) 1988-11-15

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